The objective of this study is to quantitatively understand freeze/thaw (F/T)-induced spatiotemporal cell-fluid matrix interaction during cryopreservation, and its impact on the post-thaw ECM microstructure and tissue functionality. To achieve this objective, it is hypothesized that F/T-induced cell-fluid-matrix interaction causes both spatial and temporal redistribution of interstitial fluid, which results in the post-thaw structural alteration of ECM, and this interaction can be controlled to minimize the damage on the ECM microstructure and functionality. This hypothesis will be tested by achieving the following three specific aims. Specific Aim 1. Characterize and quantify the F/T-induced cell-fluid-matrix interaction: F/T-induced spatiotemporal cell-fluid-matrix interaction will be quantitatively investigated with dermal equivalent (i.e., fibroblasts in type I collagen matrix) as a model system using a newly developed quantum dot mediated micro-/meso-scale deformetry method. Local deformation, stress, porosity and interstitial fluid pressure will be dynamically measured and analyzed during F/T. The effects of tissue dependent parameters on this interaction will be investigated. In addition, a theoretical model will be developed to simulate the interaction. Specific Aim 2. Characterize and quantify the post-thaw ECM microstructure and functional properties: Post thaw microstructure of ECM will be assessed by scanning electron microscopy and quantitatively analyzed for structural changes (i.e., pore diameter, collagen fiber diameter, and network morphology). As well as the microstructural changes, macroscopic functional properties will also be assessed before and after F/T, and correlated to the extent of the structural changes. These properties are - 1) mechanical properties including elastic modulus, creep and stress relaxation;and 2) transport properties including diffusion coefficients, tortuosity and oxygen permeability. Specific Aim 3. Characterize and facilitate post-thaw cell-ECM interaction: This SA focuses on the consequences of cryopreservation for fibroblast physiology in 3-dimensional collagen matrices, especially cell-ECM interaction. Using several different collagen matrix models, key features of fibroblast function will be measured. Floating collagen matrix remodeling tests fibroblast mechanoregulation;restrained matrix contraction tests fibroblast biosynthetic activity, differentiation into myofibroblasts and contraction;and nested collagen matrices tests fibroblast migration. Experiments will be preformed using these models to examine the effects of F/T conditions and post-thaw ECM microstructure with the goal to maximize the ability of cells to exhibit normal physiological behavior. Public Health Relevance Statement (provided by applicant): The proposed research activities have significant societal impacts including improving human health by providing easy access to engineered tissues, and providing the tissue engineering and regenerative medicine community with cost-effective method to store and bank engineered tissues.